This study evaluated a potential sterilization process that uses calcium hypochlorite (Ca(ClO)2) as a disinfectant and hydrogen peroxide (H2O2) as a neutralizing agent for monoculture processes of microalgae (Nannochloropsis oculata). The results showed that no contaminants (prokaryote) were present when the Ca(ClO)2 concentration was greater than 0.010%. The use of an equivalent amount of H2O2 completely neutralized Ca(ClO)2 and had an additional bactericidal effect because of the formation of singlet oxygen. No substantial difference was observed in the biomass accumulation and chlorophyll contents compared to those in cultures sterilized using conventional physical methods such as autoclaving. Therefore, chemical sterilization using Ca(ClO)2 and H2O2 has an excellent economic advantage, and we expect the proposed ecofriendly chemical sterilization method to become a critical culture technology for microalgae-related industrialization.